發(fā)布時(shí)間：2018-01-11 12:19

  本文關(guān)鍵詞：辣椒素對(duì)高脂飲食誘導(dǎo)肥胖小鼠腸道菌群的影響研究　出處：《第三軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 辣椒素 高脂飲食誘導(dǎo)肥胖 腸道菌群 Akkermansia 生物信息學(xué)

【摘要】：背景:肥胖癥是由遺傳因素和環(huán)境因素共同作用導(dǎo)致的營(yíng)養(yǎng)代謝障礙性疾病,可以增加代謝性疾病、心血管等疾病的發(fā)病率。現(xiàn)有的肥胖治療手段包括飲食調(diào)節(jié)/干預(yù)、運(yùn)動(dòng)鍛煉、手術(shù)減肥,但這些方法都存在耐受性差、副作用大或治療后反彈等缺點(diǎn),亟待開發(fā)新的肥胖預(yù)防和治療方法。近年來,幾種來源于天然植物的生物分子被證實(shí)具有減肥的作用,包括辣椒素,低聚果糖,多酚,巖藻依聚糖等。辣椒作為蔬菜或調(diào)味品在全球范圍內(nèi)作被廣泛食用,其主要成分辣椒素(Capsaicin)在人體和動(dòng)物上的減肥作用已經(jīng)被廣泛的報(bào)道。其中涉及到的機(jī)制較為復(fù)雜,包括全身性的機(jī)體代謝調(diào)節(jié),食物攝入量減少等。研究發(fā)現(xiàn)辣椒素激活瞬時(shí)受體電位香草醛亞家族1(Transient Receptor Potential Vanilloid 1,簡(jiǎn)稱TRPV1)鈣離子通道并增強(qiáng)腎上腺髓質(zhì)中的兒茶酚胺分泌,從而增強(qiáng)產(chǎn)熱作用起到減少體重增加的作用。研究發(fā)現(xiàn)辣椒素能減少食物攝入,且這種作用依賴于TRPV1。越來越多的證據(jù)顯示腸道微生態(tài)失調(diào)在肥胖和其它代謝性綜合癥中扮演了重要的角色。腸道微生物與宿主之間存在大量的生理學(xué)上和病理學(xué)上的相互作用,比如宿主能量平衡的調(diào)節(jié)、糖代謝、慢性炎癥狀態(tài)。研究發(fā)現(xiàn)肥胖人群和肥胖小鼠中腸道微生物群落組成發(fā)生了巨大的改變。辣椒素通過TRPV1的減肥作用已經(jīng)被廣泛研究,但是辣椒素對(duì)腸道微生物群落(腸道菌群)的影響尚未見系統(tǒng)報(bào)道。在微生物群落分析中,不管是基于16S r RNA基因的擴(kuò)增子測(cè)序(Amplicon sequencing)還是鳥槍法宏基因組測(cè)序(Shotgun metagenomics sequencing),不管是使用何種分析流程,其生物信息分析流程都涉及多個(gè)軟件,包括大量繁雜的步驟,包括測(cè)序原始數(shù)據(jù)質(zhì)量控制、分樣、格式轉(zhuǎn)換等等。如何準(zhǔn)確、高效地完成這些分析步驟是生物信息分析和開發(fā)研究工作者迫切需要解決的問題。目的:研究辣椒素對(duì)高脂飲食誘導(dǎo)肥胖小鼠腸道菌群的影響;針對(duì)一種或多種腸道微生物,研究探索辣椒素改變腸道菌群結(jié)構(gòu)的機(jī)制;設(shè)計(jì)開發(fā)微生物群落分析輔助工具。方法與結(jié)果:本研究分為三個(gè)部分:第一部分:辣椒素對(duì)高脂飲食誘導(dǎo)肥胖小鼠腸道菌群結(jié)構(gòu)的影響研究研究喂食辣椒素9周后高脂飲食誘導(dǎo)肥胖C57BL/6J小鼠與不喂食辣椒素的肥胖小鼠之間腸道微生物群落的變化,發(fā)現(xiàn)這種辣椒素的減肥作用與腸道微生物群落的改變相關(guān)。1、在微生物群落組成上,在門的水平,長(zhǎng)期食用辣椒素的高脂飲食肥胖小鼠腸道中變形菌門(Proteobacteria)豐度顯著降低。在屬的水平,擬桿菌屬(Bacteroides)、糞球菌屬(Coprococcus)、普氏菌屬(Prevotella)和阿克曼氏菌屬(Akkermansia)的豐度則顯著增加,且這些細(xì)菌豐度的增加均對(duì)健康有利。2、在微生物群落功能上,與帕金森疾病有關(guān)的代謝通路、與細(xì)菌毒力相關(guān)的四型分泌系統(tǒng)代謝模塊豐度降低,而有利于改善宿主糖代謝的糖酵解代謝模塊豐度增加。第二部分:辣椒素增加腸道細(xì)菌Akkermansia muciniphila豐度的機(jī)制研究1、辣椒素在體外直接刺激A.muciniphila結(jié)果顯示,辣椒素在高濃度下會(huì)抑制A.muciniphila的生長(zhǎng),但在低濃度下也不會(huì)促進(jìn)其生長(zhǎng);在黏蛋白(Mucin)存在下,A.muciniphila生長(zhǎng)速度提高,在平臺(tái)期細(xì)菌密度更高。2、脂餐誘導(dǎo)肥胖C57BL/6J小鼠喂食9周辣椒素后與不喂食辣椒素的肥胖小鼠相比,沒有增加腸道杯狀細(xì)胞的數(shù)量和密度。3、給正常C57BL/6J小鼠灌食辣椒素30分鐘后,空腸杯狀細(xì)胞主要黏蛋白基因Muc2表達(dá)上調(diào),并持續(xù)到90分鐘;腸道抗菌肽基因Reg3g的表達(dá)在結(jié)腸中穩(wěn)步上升。第三部分:設(shè)計(jì)開發(fā)微生物群落分析輔助工具應(yīng)對(duì)微生物群落分析的可重復(fù)性、多樣性和高效性要求,用Go語言開發(fā)多個(gè)微生物群落分析輔助工具,提供針對(duì)Windows,Linux,OS X,Free BSD,Open BSD等主流操作系統(tǒng)的單一可執(zhí)行二進(jìn)制文件,無需安裝和配置,下載即用,極大降低微生物和生物信息工作者的安裝和學(xué)習(xí)成本,已受到生物信息和開源社區(qū)廣大用戶的歡迎和認(rèn)可。其中Seq Kit和Taxon Kit均被邀請(qǐng)錄入英文書籍《The Biostar Handbook:A Beginner's Guide to Bioinformatics》中。1、設(shè)計(jì)、開發(fā)、高度優(yōu)化FASTA/Q序列處理軟件包Seq Kit,提供20個(gè)實(shí)用的子命令和豐富的文檔,提高測(cè)序數(shù)據(jù)的處理效率。2、設(shè)計(jì)、開發(fā)CSV/TSV格式處理軟件包c(diǎn)svtk,提供25個(gè)易用和實(shí)用的子命令,用于處理微生物群落分析中的數(shù)據(jù)文件。3、設(shè)計(jì)、開發(fā)NCBI分類學(xué)數(shù)據(jù)處理軟件包Taxon Kit,用于高效查詢、轉(zhuǎn)換NCBI生物分類學(xué)數(shù)據(jù),用于應(yīng)對(duì)不同微生物群落分析軟件的格式需求。4、設(shè)計(jì)、開發(fā)作業(yè)并行軟件rush,提供高實(shí)用性的功能,用于簡(jiǎn)化和加速對(duì)大量處理樣品的包含微生物群落分析在內(nèi)的生物信息分析。結(jié)論:1、高脂飲食誘導(dǎo)肥胖小鼠長(zhǎng)期食用辣椒素能減輕體重,改善葡萄糖穩(wěn)態(tài)。這種改善作用與腸道菌群的改變有關(guān)。腸道菌群的改變及腸道細(xì)菌Akkermansia muciniphila的豐度增加可能在這種保護(hù)性作用中起到關(guān)鍵作用。因此我們推測(cè)辣椒素可能通過改變腸道菌群從而起到減肥作用。2、辣椒素增加A.muciniphila的可能機(jī)制包括:(1)辣椒素刺激杯狀細(xì)胞分泌更多的黏蛋白,為其提供更多的食物來源,從而使得A.muciniphila絕對(duì)豐度增加;(2)腸道抗菌蛋白R(shí)eg3g分泌增加,抑制其它細(xì)菌,從而使得A.muciniphila相對(duì)豐度增加;(3)小鼠食物攝入量減少,其它細(xì)菌因食物來源減少而豐度降低,而A.muciniphila可以以黏蛋白作為唯一的氮源和氮源,不受影響從而相對(duì)豐度增加。3、本研究設(shè)計(jì)開發(fā)多個(gè)跨平臺(tái)、易用、實(shí)用、高效的微生物群落分析輔助軟件,以應(yīng)對(duì)微生物群落分析的可重復(fù)性、多樣性和高效性要求,滿足日益增長(zhǎng)的測(cè)序大數(shù)據(jù)的分析需求。FASTA/Q格式處理工具Seq Kit、CSV/TSV格式處理軟件包c(diǎn)svtk、NCBI分類學(xué)數(shù)據(jù)處理軟件Taxon Kit和作業(yè)并行軟件rush能極大提高微生物和生物信息工作者的效率,簡(jiǎn)化和加速對(duì)大量樣品的包含微生物群落分析在內(nèi)的生物信息分析,已受到廣大用戶的歡迎和好評(píng)。
[Abstract]:Background: obesity is a nutritional and metabolic disorder caused by genetic and environmental factors that can increase metabolic diseases, the incidence of cardiovascular disease. The existing obesity treatment methods including diet / intervention, exercise, weight loss surgery, but these methods have poor tolerance, side effects or after treatment, the rebound and other shortcomings, the need to develop new methods of prevention and treatment of obesity. In recent years, several biological molecules derived from natural plants was confirmed to have a slimming effect, including capsaicin, fructooligosaccharides, polyphenols, chitosan and so on. According to the rock algae as a vegetable or chili seasoning in global range is widely consumed, the main ingredient capsaicin (Capsaicin) in human and animal weight loss has been widely reported. The mechanism involves more complex, including systemic metabolic regulation of food intake. Reduced. Study found that capsaicin activates transient receptor potential subfamily (Transient Receptor 1 with Potential Vanilloid 1, referred to as TRPV1) calcium channel and increase catecholamine secretion in the adrenal medulla, thereby enhancing thermogenesis to reduce weight gain for study found that capsaicin can reduce food intake, and this effect is dependent on TRPV1. more and more evidences show that intestinal microflora imbalance plays an important role in obesity and other metabolic syndrome. There are a large number of physiological and pathological studies on the interaction between gut microbiota and host host, such as the regulation of energy balance and glucose metabolism and chronic inflammation. The study found that intestinal microflora of obese people and in the composition of obese mice changed. Capsaicin effect of TRPV1 by weight loss has been widely studied, but the chili The intestinal microbial community (gut flora) effects have not been reported. In the analysis of microbial community, whether amplicon sequencing 16S R gene based on RNA (Amplicon sequencing) or shotgun metagenome sequencing (Shotgun metagenomics sequencing), which is not the analysis process, the biological information analysis process involves a number of software, including a large number of complex steps, including the original sequencing data quality control, sample, format conversion and so on. How to accurately and efficiently complete the analysis steps as researchers of biological information analysis and development of the urgent problem to be solved. Objective: To study the effect of capsaicin on high fat diet induced obesity affects the intestinal flora of mice; according to one or several microorganisms, exploring the change mechanism of capsaicin of intestinal microflora; design and development of microbial community analysis tool. Methods and results: This study is divided into three parts: the first part: the changes of intestinal microbial community between capsaicin on high fat diet induced obese mice intestinal flora structure influence research on the feeding of capsaicin after 9 weeks of high fat diet induced obese C57BL/6J mice and obese mice fed capsaicin, capsaicin found that the weight loss effect and the intestinal microbial community is related to the change of.1 in the microbial community composition, on the level of mouse intestinal bacteria door deformation of high fat diet and long-term consumption of capsaicin (Proteobacteria) was significantly lower in abundance. At the genus level, Bacteroides (Bacteroides), coprococcus (Coprococcus), P. Copri (Prevotella) and Ackerman's genus (Akkermansia) abundance was significantly increased, and the increase of the bacterial abundance are beneficial to health in.2, microbial community function, associated with Parkinson disease The metabolic pathways associated with bacterial virulence, type four secretion system metabolic module abundance was reduced, and is conducive to the improvement of glucose metabolism of host glycolytic module abundance increased. The second part: the mechanism of capsaicin increased intestinal bacteria Akkermansia muciniphila abundance of 1 capsaicin in vitro direct stimulation of A.muciniphila results showed that capsaicin could inhibit A.muciniphila in under the high concentration of growth, but at low concentrations will not promote its growth; mucin (Mucin) in the presence of A.muciniphila growth rate increase in plateau bacterial density higher.2, compared with the fat meal induced obese C57BL/6J mice were fed 9 weeks after feeding and capsaicin capsaicin in obese mice, no increase in intestinal goblet the cell number and density of.3 to normal C57BL/6J mice fed capsaicin after 30 minutes, jejunal goblet cells mainly mucin gene expression of Muc2 was up-regulated, and Continued to 90 minutes; the expression of intestinal antibacterial peptide gene Reg3g increased steadily in the colon. The third part: the analysis of auxiliary tools to cope with the microbial community reproducibility of the design and development of microbial community diversity and efficiency requirements, the development of a microbial community analysis tool using Go language, for Windows, Linux, OS X, Free BSD, Open BSD and other mainstream operating system of the single executable binary file, no need to install and configure, download and use, greatly reducing the microbial and biological information workers and the installation cost of the study, has received the biological information and the open source community recognized and welcomed by the customers. The Seq Kit and Taxon Kit were .1, design, development, highly optimized FASTA/Q sequence processing software package Seq Kit, provides 20 practical sub commands And the rich design documents, improve the processing efficiency of.2 sequencing data, the development of CSV/TSV format processing software package csvtk 25 is easy to use and practical command, for the treatment of microbial community analysis in.3 data files, design, development of NCBI taxonomy data processing software package Taxon Kit, used for efficient query conversion, NCBI taxonomic data, used to deal with different microbial community analysis software format requirements of.4 design, development, operation of parallel software rush, providing high availability features, for the analysis of biological information to simplify and accelerate the amount of processing samples containing microbial community analysis,. Conclusion: 1, long-term consumption of capsaicin can reduce the body weight induced by high fat diet obese mice, improve the glucose homeostasis. The improvement of intestinal flora and changes. The abundance of intestinal flora and intestinal bacteria Akkermansia muciniphila. May play a key role in the protective effect. So we speculate that capsaicin may alter the intestinal flora and thus play a slimming effect including.2, capsaicin increased the possible mechanism of A.muciniphila (1): capsaicin stimulates secretion of mucin more goblet cells, provide more sources of food, which makes the A.muciniphila absolute abundance increased; (2) intestinal Reg3g protein secretion, inhibition of other bacteria, so that the relative abundance of A.muciniphila increased; (3) to reduce food intake in mice, because other bacterial food source and reduce the abundance decreased, while A.muciniphila with mucin as nitrogen source and nitrogen source only, which is not affected by the increase in relative abundance.3, the research design and development of multiple cross platform, easy to use, practical, efficient microbial community analysis software, to deal with the microbial community analysis repeatability, Diversity and efficiency requirements, meet the growing of the sequencing data analysis needs.FASTA/Q format processing tool of Seq Kit, CSV/TSV format software package csvtk, NCBI taxonomy data processing software Taxon Kit and rush parallel software can greatly improved the efficiency of microbial and biological information workers, including simplified and accelerated biological information analysis of microbial community the analysis of a large number of samples, and has been welcomed by the majority of users and praise.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R589.2

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